Extracellular Peptide-Ligand Dimerization Actuator Receptor Design for Reversible and Spatially Dosed 3D Cell-Material Communication

Transmembrane receptors
that endow mammalian cells with the ability
to sense and respond to biomaterial-bound ligands will prove instrumental
in bridging the fields of synthetic biology and biomaterials. Materials
formed with thiol-norbornene chemistry are amenable to thiol-peptide
patterning, and this study reports the rational design of synthetic
receptors that reversibly activate cellular responses based on peptide-ligand
recognition. This transmembrane receptor platform, termed Extracellular
Peptide-ligand Dimerization Actuator (EPDA), consists of stimulatory
or inhibitory receptor pairs that come together upon extracellular
peptide dimer binding with corresponding monobody receptors. Intracellularly, Stimulatory EPDAs phosphorylate a substrate that merges
two protein halves, whereas Inhibitory EPDAs revert
split proteins back to their unmerged, inactive state via substrate
dephosphorylation. To identify ligand-receptor pairs, over 2000 candidate
monobodies were built in silico using PETEI, a novel
computational algorithm we developed. The top 30 monobodies based
on predicted peptide binding affinity were tested experimentally,
and monobodies that induced the highest change in protein merging
(green fluorescent protein, GFP) were incorporated in the final EPDA
receptor design. In soluble form, stimulatory peptides induce intracellular
GFP merging in a time- and concentration-dependent manner, and varying
levels of green fluorescence were observed based on stimulatory and
inhibitory peptide-ligand dosing. EPDA-programmed cells encapsulated
in thiol-norbornene hydrogels patterned with stimulatory and inhibitory
domains exhibited 3D activation or deactivation based on their location
within peptide-patterned hydrogels. EPDA receptors can recognize a
myriad of peptide-ligands bound to 3D materials, can reversibly induce
cellular responses beyond fluorescence, and are widely applicable
in biological research and regenerative medicine.